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  • In this next step, we will transfer our separated proteins out of the gel and

  • into a solid membrane or blot.

  • This is based upon the same principal as the previous step in which an electric

  • field is charged to remove the negative proteins towards a positive

  • electrode.

  • Transfer can occur under wet or semi-dry conditions. Here we will demonstrate the

  • traditional wet transfer method. Start by removing the gel from its cassette

  • cutting the top portion containing the wells.

  • Notch the top left corner to indicated gel orientation.

  • Float the gel in transfer buffer while preparing the transfer sandwich.

  • To make the transfer sandwich you will need a cassette,

  • sponge, filter paper,

  • the gel

  • gel and your choice of either PVDF

  • or nitrocellulous membrane.

  • PVDF must first be activated by soaking the membrane in ethanol for

  • two minutes.

  • But other than this the PVDF or choice of nitrocellulous

  • membrane is a personal preference.

  • Notch the top left corner to indicate blot orientation

  • and incubate membranes in transfer buffer for 10 minutes.

  • Create a stack by placing the following components from the black

  • negative cathode to red positive anode:

  • sponge,

  • filter paper, gel,

  • membrane.

  • (Be careful not to touch the gel or membrane with your bare hands and

  • use clean tweezers or spatula instead.

  • Touching the membrane during any phase can contaminate the blot and lead to

  • excessive background signal.)

  • filter paper

  • and sponge.

  • Use a clean roller with each layer to gently roll out any bubbles that may be

  • present since bubbles will inhibit efficient protein transfer.

  • Lock the cassette and place it in the apparatus containing cold

  • transfer buffer

  • ensuring that the cassette is properly positioned from negative to positive.

  • In order to prevent heat buildup, it is beneficial to transfer with a cold

  • pack in the apparatus or in a cold room with the spinner bar placed at the

  • bottom of the chamber.

  • Close the chamber and connect to a power supply.

  • Perform the transfer according to the manufacturer's instructions which is

  • normally 100 volts for thirty to one hundred and twenty minutes.

In this next step, we will transfer our separated proteins out of the gel and

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